Cytoplasmic Damage and Genotoxicity

描述（由申请人提供）：自Rventgen发现x射线以来的一个多世纪以来，放射科学专业的学生一直被教导说，辐射引起的遗传损伤的典型目标存在于细胞核的DNA中。细胞质损伤的生物学后果在很大程度上是未知的。申请人使用精密带电粒子微束和双荧光染料分别定位细胞核和细胞质，从而避免了细胞核的无意穿越，先前已经表明，细胞质辐照实际上在人-仓鼠杂交（AL）细胞的CD59位点具有诱变作用，同时造成最小的细胞毒性。在母体R01的资助下，我们最近发现：1)活性氧介导了这一过程；2)靶向细胞质照射导致细胞膜脂质过氧化，通过诱导脂质过氧化的主要副产物4-羟基-2-壬烯醛（4-羟基-2-壬烯醛）表现出来；3)生成线粒体DNA缺失（Ao） AL细胞和人成纤维细胞；最后，靶向细胞质照射通过间隙连接介导的过程在邻近的非照射细胞中诱导旁观者突变和染色单体断裂。这就提出了以下问题：靶向细胞质辐照对线粒体DNA突变的影响是什么？线粒体DNA缺失的人成纤维细胞在细胞质辐照下是否表现出减少诱变诱导？这项为期一年的补充应用的中心假设是，靶向细胞质照射诱导人成纤维细胞线粒体DNA的剂量依赖性突变，线粒体DNA缺失的细胞对基因毒性信号的反应较差。为了解决这些假设，提出了两个具体目标。突变将在人类成纤维细胞的HPRT位点和线粒体DNA突变（缺失、异质缺失和点突变）上进行评分。提出的研究将有助于解决细胞质辐照如何导致细胞核遗传事件的机制。与旁观者诱变效应一起，该研究将解决一些关于核外靶点和细胞质损伤如何在哺乳动物细胞中处理的基本问题。

DESCRIPTION (provided by applicant): For over a century since the discovery of X-rays by Rventgen, students in radiological sciences have been taught that the quintessential target for radiation induced genetic damages resides in the DNA of the nucleus. The biological consequences of cytoplasmic damage are largely unknown. Using a precision charged particle microbeam and dual fluorochrome dyes to locate nucleus and cellular cytoplasm respectively, thereby avoiding inadvertent traversal of nuclei, the applicant has shown previously that cytoplasmic irradiation is, in fact, mutagenic at the CD59 locus of human-hamster hybrid (AL) cells while inflicting minimal cytotoxicity. With the funding support of the parent R01, we have shown recently that 1) reactive oxygen species mediate this process; 2) targeted cytoplasmic irradiation results in lipid peroxidation of membranes as shown by the induction of 4-hdyroxy-2-nonenal, a major lipid peroxidation byproduct; 3) generated mitochondrial DNA depleted (Ao) AL cells and human fibroblasts; and finally, targeted cytoplasmic irradiation induces bystander mutations and chromatid breaks among neighboring, non-irradiate cells through a gap junction-mediated process. This raised the following questions: What are the effects of targeted cytoplasmic irradiation on mitochondrial DNA mutations and does mitochondrial DNA depleted human fibroblasts demonstrated reduced induction of mutagenesis upon cytoplasmic irradiation? The central hypotheses for this one year supplemental application is targeted cytoplasmic irradiation induces a dose dependent mutagenesis of mitochondrial DNA in human fibroblasts and that mitochondrial DNA depleted cells respond poorly to genotoxic signaling. To address these hypotheses, 2 specific aims are proposed. Mutations will be scored at the HPRT locus in human fibroblasts and mitochondrial DNA mutations (depletion, heteroplasmic deletions and point mutations will be determined. The proposed studies will help to address the mechanisms of how cytoplasmic irradiation results in a genetic event in the nucleus. Together with the bystander mutagenic effect, the study will address some of the fundamental issues regarding extranuclear target and how cytoplasmic damages are being processed in mammalian cells. PUBLIC HEALTH RELEVANCE: Generations of students in radiation biology have been taught that heritable biological effects induced by ionizing radiation are the consequence of a direct radiation-nuclear interaction. Using the Columbia University charged particle microbeam and the human hamster hybrid (AL) cell mutagenic assay, there is evidence that targeted cytoplasmic irradiation is mutagenic in mammalian cells. This first, unequivocal demonstration of an extranuclear effect of ionizing radiation provides strong support of the subsequent, broad reaching bystander/ non-targeted effects of radiation. There is evidence that targeted cytoplasmic irradiation can also induce a bystander effect. As such, a better understanding of the genotoxic mechanism of cytoplasmic irradiation is critical in low dose radiation risk assessment. Mitochondria are widely distributed in the cytosol and mitochondrial DNA is highly susceptible to oxidative damage. The objective of the proposed study is to ascertain the possible effects of cytoplasmic irradiation on mitochondrial DNA mutations and the subsequent modulation on mitochondrial function. The study will address some of the fundamental issues regarding extranuclear target and how cytoplasmic damages are being processed in mammalian cells.